Project 1. Defining upstream and downstream components of Nodal signaling in gastrulation. [unreadable] [unreadable] Modulators of sqt penetrance. Background: cyc embryos have a more limited mesoderm and endoderm deficit than sqt;cyc double-mutant embryos, namely a reduction in anterior axial mesoderm and endoderm, leading to holoprosencephaly (HPE). This phenotype is seen in only a fraction of sqt mutants, with many appearing wild type. Here at NHGRI, we examined the basis of sqt phenotypic variability and found that co-inherited genes, environmental temperature and Heat Shock Protein 90 function all influence HPE incidence in these mutants. Over the previous 12 months, we performed a number of control experiments to support and extend our previous conclusions, leading to the publication of a manuscript in the journal Developmental Biology in 2007. Our latest work has focussed on testing the hypothesis that this represents a genetic interaction of the Nodal gene Squint with Wnt pathway genes involved in planar cell polarity.[unreadable] [unreadable] Function of FoxH1. Background: One challenge to dissecting the Nodal signaling pathway is the diversity of phenotypes resulting from perturbations of FoxH1 function. At NHGRI, we have characterized zebrafish embryos treated with antisense morpholino oligonucleotides (MOs) designed to block translation of maternal and zygotic FoxH1 protein. These embryos display a striking down regulation of keratin genes and dramatic disruptions in cell movement. This phenotype, similar to antisense FoxH1 disruptions in Xenopus laevis, suggests that FoxH1 has Nodal-independent functions. By contrast, a milder phenotyope is seen in the zebrafish schmalspur (sur) mutants, two FoxH1 alleles with point mutations in the winged-helix DNA binding domain. We have accordingly hypothesized that the severe phenotype associated with FoxH1 knockdown is due to the loss of FoxH1 functions outside of the DNA-binding domain. Over the previous 12 months, we performed a number of control experiments to support and extend our previous conclusions, leading to the publication of a manuscript in the journal Developmental Biology in 2007. To test the hypothesis that FoxH1 domains outside of the DNA-binding domain have Nodal-independent functions, we have most recently been seeking to identify new lines of zebrafish carrying mutations in the foxh1 gene.[unreadable] [unreadable] [unreadable] Project 2. Identifying roles of RhoGTPase signaling in gastrulation movements. [unreadable] Background: RhoGEF proteins are positive regulators of RhoGTPases, which have profound roles in cellular movement and morphology. To identify RhoGEFs with roles in directing the morphogenetic events of gastrulation, we performed a loss-of-function screen. We identified 48 RhoGEFs expressed during early embryogenesis and determined the loss-of-function phenotypes for 23 of these, using a non-invasive embryo holding system we designed that allows for the parallel time-lapse documention of 54 embryos. We thus identified five RhoGEFs for which two independent MOs produced the same phenotype. Three of these, homologues of ARHGEF16, Frabin and Net1, respectively, disrupted epiboly. Two others, ARHGEF10 and PLEKHG4 homologues, caused post-gastrulation defects during somitogenesis stages. During the last 12 months, we have been looking for and testing new lines of zebrafish carrying mutations in these genes. [unreadable] [unreadable] Project 3. Defining roles of blastula margin-enriched proteins and behaviors of blastula margin cells. [unreadable] Background: A precise separation of newly-specified germ layer precursors in zebrafish embryos, has been untenable using conventional tools. Over previous funding years we have developed and refined a novel technique for precisely microdissecting embryonic regions of interest: FACS-assisted microdissection of photolabeled cells (FAM-P). We used this method to separate mesoderm and endoderm (mesendoderm) precursor cells from ectoderm precursor cells of pre-gastrula stage embryos, and used microarrays to assess their respective transcriptomes. Over the last 12 months, we have compared the behaviors of mesendoderm and ectoderm precursors in transplantation assays and in cell cycle assays. This has shown us that both populations retain their documented properties, and has revealed cell cycle differences between these two populations. We also performed a detailed analysis of the microarray data from the previous funding period, revealing that mesendoderm precursor cells express fewer enzyme-encoding genes and demonstrating that FAM-P purification effectively depletes extra-embryonic cells. Finally, we have investigated the requirements of several mesendoderm precursor-specific genes identified in these studies, using antisense reagents. One of these, dusp4, is required for normal head development and the differentiation of late endoderm.